October 26, 2006 1
Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell 1. Secretory pathway a. Formation of coated vesicles b. SNAREs and vesicle targeting 2. Membrane fusion a. SNAREs and vesicle fusion 3. Retrograde trafficking a. Steady-state 4. Functions of the Golgi a. Glycosylation b. Protein sorting 5. Viral assembly and exit from the cell Lecture Readings Alberts 498-522 (cont.) 2
Challenges in Vesicular Transport I 1) How is membrane deformed to form a vesicle? 2) How are the correct cargoes enriched in the vesicle and the proteins that should remain in the donor compartment excluded? 3) How do vesicles know what compartment to fuse with? 4) What catalyzes the fusion process? The use of vesicles to move proteins and lipids from one compartment to another raises several challenges that must be overcome. For example, the membrane must be deformed to make a vesicle and this process is energetically unfavorable because the head groups at the neck of the budding vesicle are brought close together, resulting in unfavorable electrostatic interactions. Additionally, the appropriate cargoes must be packaged into vesicles and the proteins that are to remain in the donor compartment must be left behind. Once vesicles are formed, there has to be a mechanism that allows vesicles to know what compartment to fuse with. Finally, as we saw with HIV entry into cells, membrane fusion is energetically unfavorable. In that case, energy stored in the structure of gp41 was used to drive the fusion process. There has to be an analogous mechanism operating for the fusion of vesicles with target membranes in the cell. 3
3 Major Types of Coated Vesicles Proteins coating vesicles that bud from donor compartments play a critical role in addressing the first two questions - vesicle formation and cargo selection. Vesicles bud off as coated vesicles that have a cage of proteins covering their cytosolic surface. There are three major types of coated vesicles: clathrin, COPI, and COPII. The coats have two major functions: (1) deforming membrane/introducing curvature to form a vesicle Formation of a vesicle requires curvature of the membrane, which brings negatively charged phospholipids into close proximity in places, an energetically unfavorable act. Introduction of curvature is achieved in part by neutralizing the negative charge on the phospholipid head groups, making it more favorable for the membrane to be deformed. Curvature is also introduced by the shape of the coat proteins themselves; some are known to have intrinsic curvature and to polymerize to form a partial sphere. The tight binding of the coat protein to the membrane and coat protein polymerization causes the membrane to deform. (2) concentrating/selecting appropriate cargo Coats accomplish this by interacting with proteins that are to be enriched in the vesicle. These interactions may be direct or may be mediated through other proteins. Coats are used for different transport steps in the cell: Clathrin - movement of proteins from the Golgi and plasma membrane. COPI and COPII - movement of proteins between the Golgi and the ER. 4
SNAREs and Targeting Vesicles coat protein Vesicles have to recognize the correct target membrane; because there are many membranes, a vesicle is likely to encounter many potential targets. Proteins called SNAREs (which stands for Golgi SNAP receptor complex protein, a name that is not particularly informative) are thought to play a role in determining which vesicles fuse with which compartments. Specifying in targeting is ensured because vesicles display v-snares (vesicle SNAREs) on their surface that identify them according to their origin and type of cargo; target membranes display complementary t-snares (target SNAREs) that recognize the v-snares. For example, in this picture the skin-colored v-snare pairs with the light blue t-snare on compartment A, targeting cargo a to compartment A. Similarly, the red v-snare on vesicles containing cargo b binds to the bright blue t-snare on compartment B. This diagram also shows the cargo receptor (brown) and vesicle coat (green); we previously discussed how the coat proteins play a role in deforming the membrane to form a vesicle and in helping to select cargo for inclusion in the vesicle. Cargo selection is achieved indirectly - the coat protein interacts with a cargo receptor, which in turn binds to cargo proteins (yellow) that are to be included in the vesicle. These interactions ensure that the correct cargo is selected for inclusion in the vesicle. 5
SNAREs Promote Membrane Fusion v-snare vesicle SQUEEZE OUT WATER FORM STALK target t-snare HEMI- FUSION FUSION In addition to their role in targeting vesicles to the correct target compartments, SNAREs are thought to play a role in membrane fusion. The role of SNAREs is analogous to the role played by HIV Gp41 in fusion of the virus with the cell membrane. Recall that in the case of Gp41, the favorable change in Gp41 structure is used to overcome the unfavorable electrostatic repulsion that occurs when the viral and host cell membranes are brought together. v-snares pair with t-snares to form a very stable bundle of four alpha-helices that forces the two membranes into close apposition; like Gp41, the favorable energetics of folding (into a bundle of four alpha-helices) is used to overcome the unfavorable process of expelling water molecules from the interface and bringing charged lipid head groups together (1.5 nm separation of the two membranes). The lipids of the two interacting leaflets then flow between the membranes to form a connecting stalk. Next, the lipids of two other leaflets contact each other, forming a new bilayer which widens the fusion zone (hemifusion state). Finally, rupture of the new bilayer completes fusion of vesicle and target. 6
Challenges in Vesicular Transport II 1) How does the cell maintain organelles and the plasma membrane at a constant size if vesicles are constantly moving lipids from compartments to the plasma membrane? 2) How does the cell ensure that proteins which should remain in a compartment do so, even if the process of cargo selection is not perfect? We talked about how proteins get into the secretory pathway by crossing the ER membrane and how proteins leaving the ER move in vesicles which fuse with the Golgi membrane to deliver their contents. This raises two questions regarding the transport and its consequences. First, how does the cell maintain organelles and the plasma membrane at a constant size if vesicles are constantly moving lipids from compartments to the plasma membrane? This one way transport from the ER to the Golgi and on to the plasma membrane would tend to decrease the size of the ER and increase the size of the plasma membrane. Second, how does the cell ensure that the proteins that are to remain in a compartment do so, given that the process of cargo selection is not perfect? 7
Secretory Pathway: Retrograde Traffic nuclear envelope endoplasmic reticulum lysosome late endosome plasma membrane early endosome CYTOSOL cisternae Golgi apparatus secretory vesicle The answer to both questions lies in the process of retrograde traffic - membrane vesicles move back from plasma membrane into the secretory pathway and from the Golgi to the ER (green arrows) - this provides an opportunity for proteins that were mis-sorted to be sent back to the correct compartment; retrograde traffic also ensures that the balance of lipids and the relative size of the compartments is maintained. Flow of lipids in the secretory pathway is at steady-state. This is true of almost all processes in a living cell - they are steady-state, not at equilibrium. 8
Equilibrium vs. Steady-State equilibrium steady-state What is steady-state and what is the difference between steady-state and equilibrium? Steady-state is similar to equilibrium, but not the same. In steady-state, the system is stable (unchanging), and energy has to be put into the system in order to maintain it. An example of steady-state is the potassium ion concentration within cells - the concentration is stable, but maintenance of this stable state requires the input of ATP to the sodium-potassium pump. In equilibrium, the system is also stable. However, no energy has to be put into the system to maintain this stable state. For example, the ionization of HCl in a beaker of water is a system that quickly reaches equilibrium where HCl is ionized and the system is at its lowest free energy state. This happens without the input of any energy. A simple way to think about this is to consider a bathtub: if the bathtub doesn't leak (left), your bathwater will stay at the same level indefinitely at equilibrium. But if the bathtub does leak, the water level will drop at some rate (middle right). By matching the rate with which you add water (top right) to the rate with which you lose water (middle right), you can achieve a constant, non-equilibrium steady-state (bottom right). Trafficking of vesicles in the secretory pathway is at steady-state - the size of the ER is not changing, even though vesicles are budding and fusing - the rate vesicles bud from the ER is approximately equal to the rate they fuse with it. Since there are net movements of molecules and energy is expended during trafficking, the system is not at equilibrium. Processes in the cell are generally far from equilibrium, but some are at steady-state. Exception: Binding reactions within cells can be at equilibrium. 9
Golgi Apparatus vesicles coming from ER vesicles leaving Golgi Vesicles leaving the ER fuse with the Golgi, a collection of flattened membrane-enclosed sacs called cisternae. Coated vesicles from the ER fuse with the cis-face of the Golgi network, move through the cisternae, and exit from the trans-face of the Golgi network where they are directed to various compartments within the cell. The accepted model for transport through the Golgi is called the cisternal maturation model. It postulates that each cisterna is a transient structure that matures from cis to trans by acquiring and then losing specific Golgi-resident proteins. In this view, secretory cargo proteins traverse the Golgi by remaining within the maturing cisternae. The major functions of the Golgi include: (1) Sorting proteins to different destinations Proteins destined for the endosome, plasma membrane, or the ER are sorted in the Golgi and targeted to those compartments; retrograde traffic of proteins containing ER retention signals sends proteins from the Golgi to the ER and ensures that the ER retains its complement of proteins. (2) Glycosylation Carbohydrates are further processed - added, modified and removed - in the Golgi apparatus. This is an important step in the production of mature glycoproteins such as HIV gp120 and gp41. 10
Viral Entry and Integration reverse transcriptase plasma membrane RNA-DNA hybrid DNA chromosomal DNA viral RNA NUCLEUS Now that we know about eukaryotic cell structure and vesicle trafficking, let s review the viral life cycle and then go back to the question of how the virus assembles a membrane and glycoprotein coat and exits the cell. To remind you, the viral coat proteins gp41 and gp120 play a crucial role in targeting HIV to its host cells, macrophages and T cells. Gp120 interacts with the CD4 protein displayed on the surface of these immune cells, and this interaction triggers a change in the gp120 structure so that it is now able to also bind to the co-receptor. Co-receptor binding triggers a structural change that releases the buried fusion peptide in the HIV fusion protein gp41, causing it to embed the fusion peptide in the host cell membrane. A slow conformational rearrangement of gp41 into a helical bundle brings the cell and viral membranes into close apposition, triggering lipid mixing and membrane fusion. Once the membranes fuse, the viral genome and proteins are deposited into the cell. The capsid proteins (orange) are then removed, releasing viral proteins and the viral RNA. In a process Rob will describe, reverse transcriptase copies the viral RNA into DNA, and integrase catalyzes the integration of this DNA into the host chromosome. It is this DNA copy of the viral genome that is transcribed by cellular RNA polymerase to make RNA copies of the viral genome and also to make mrnas for translation into viral proteins. 11
Viral Assembly and Exit from the Cell gp120 and gp41 plasma membrane viral capsid viral genome mrna HIV viral proteins ER, Golgi, secretory pathway gp120 and gp41 virus budding gag polyprotein Once the viral mrnas are translated and new copies of the viral genome are made, the virus must assemble new viral particles with its genome inside, bound to structural proteins, encased in membrane, with glycoproteins displayed on the outside. The virus must then exit the cell. The viral capsid proteins and enzymes are made in the cytosol, but we learned that the glycoproteins gp120 and gp41 traffic through the secretory pathway and acquire carbohydrate modifications in the ER and Golgi. Once in the Golgi g120 and gp41 are routed to the plasma membrane, oriented so that the carbohydrate modifications are on the outside of the cell. The viral RNA genome is produced by transcription of a DNA copy (you will hear about this from Rob in the next lectures). A protein called Gag plays a crucial role in viral assembly. Gag is made as a polyprotein (really 4 proteins strung together) and has 4 domains: MA, modified by a lipid group and involved in targeting Gag to the plasma membrane; CA, involved in assembling the viral particle; NC, which captures the viral RNA genome; and p6, which recruits cellular proteins that help in viral release. Gag is made in the cytoplasm and modified with a lipid group, which targets it to the plasma membrane. At the membrane, it recruits the viral genome (through interactions of the viral RNA with NC). Budding of the virus is a fission event, similar in topology to the formation of coated vesicles. The viral Gag protein is thought to play a role in deforming the plasma membrane to help promote fission, releasing viral particles complete with a membrane (derived from the plasma membrane of the host cell) and glycoproteins gp41 and gp120. Note that the fission process is analogous to the process of vesicles budding from the ER - the vesicle coat proteins deform the membrane to make vesicles and Gag protein deforms the plasma membrane to allow viral particles to escape from the cell. 12
Summary of Main Points Vesicles bud as coated vesicles containing a protein coat that plays a role in membrane deformation and cargo selection SNAREs play an important role in targeting vesicles to the correct compartment SNAREs play an important role in overcoming the energy barrier to membrane fusion; they function in a manner analogous to gp41 Retrograde, or reverse, traffic in the secretory pathway ensures that resident proteins remain in their compartments and that cells maintain organelles at a constant size; trafficking in the secretory pathway is at steady-state Proteins are sorted to their destinations in the Golgi and carbohydrates are further processed HIV assembles viral particles at the plasma membrane and buds from the cell; the viral membrane is derived from the host cell plasma membrane 13